Tissue regeneration

ABSTRACT

A biocompatible, biodegradable composition for encouraging controlled growth, regeneration or repair of biological tissue or cells, the composition comprising a scaffold, formed from biodegradable and biocompatible material, and a receptor for a growth factor, or a growth factor-binding fragment or homologue thereof, located at or adjacent a surface of the scaffold. The tissue or cells are preferably neuronal, in which case the receptor is preferably a tyrosine receptor kinase (Trk), or a neurotrophin-binding fragment or homologue thereof. Such a composition may include one or more types of neurotrophin bound to the Trk or fragment or homologue thereof.

This invention relates to the regeneration and repair of biologicaltissues. In particular, although not exclusively, the invention concernsthe use of biocompatible compositions for encouraging growth,regeneration and/or repair of neuronal tissue.

Nerve repair using autograft material has several shortcomings,including donor site morbidity, inadequate return of function, andaberrant regeneration. Alternatives to autografts have been sought foruse in bridging neural gaps. Many entubulation materials have beenstudied, although with generally disappointing results in comparisonwith autografts. Recently, peripheral nerve research has focused on thegeneration of synthetic nerve guidance conduits that might overcomethese problems. In various laboratories, synthetic biodegradablepolymers, which are removed from the engineered tissues by hydrolysisand dissolution of breakdown products, have been used in conjunctionwith Schwann cells to create a superior prosthesis for the repair ofbranched peripheral nerves (Hadlock et al (1998) Arch Otolaryngol HeadNeck Surg, 124: 1081; Hadlock et al (2000) Tissue Eng, 6: 119; Bryan etal (2000) Tissue Eng, 6: 129). The functioning of tissues such as nervesand blood vessels is dependent on the controlled orientation of cells;for many tissue cell types, the spatial organisation is required toensure that cell-to-cell interactions occur. Synthetic materials may beengineered such that they mimic cellular microenvironments encounteredduring natural development. For example, biodegradable polymer surfacescan be engineered to present peptides containing the amino acid sequencearginine-glycine-aspartate (RGD). This sequence binds to integrinreceptors on cell surfaces, inducing cell adhesion, spreading andintracellular signalling, and hence mimicking cell-to-extracellularmatrix interactions.

There are a range of techniques by which biomolecules can be immobilizedon surfaces with micron-scale precision. These techniques includelithographic methods, which use patterned masks to restrict the locationof interactions between a beam of light, ions or electrons and asurface, and micro-contact printing techniques. These techniques,however, can restrict the types of ligands and surfaces that can bepatterned.

As shown in WO99/36107, it is possible to generate micron-scale patternsof biotinylated ligands on the surface of a biodegradable blockcopolymer, achieving control of biomolecule deposition with nanometerprecision. This is confirmed by molecular resolution of proteinmolecules on the patterned surfaces using atomic force microscopy. Thissystem has been tested in cultured bovine aortic endothelial cells andPC12 nerve cells and shows spatial control over cell development.Neurite extension of PC12 cells, on the polymer surface, can be directedby pattern features composed of peptides containing the IKVAV sequence(Patel et al (1998) FASEB J, 12: 1447; Cannizaro et al (1998) BiotechnolBioeng, 58: 529).

The polymer used in the above system is generally a block copolymer ofbiotinylated poly(ethylene glycol) (PEG) with poly(lactic acid) (PLA)which uses the high affinity coupling of biotin-avidin as postfabrication surface engineering. These poly(esters) are susceptible toacid catalysed hydrolysis and are thus biodegradable. Biodegradabilityrate may be controlled and thus the polymers may be used for thecontrolled delivery of therapeutic agents. The pH-sensitivity of arelated class of polymers, the poly (orthoesters), has also been studiedfor this purpose (Leadley et al (1998) Biomaterials, 19: 1353-60).

However, there is a drawback to all of the methods used previously.Neurones require, for their maintenance and neurite outgrowth, thepresence of various growth factors. Experiments carried out under cellculture conditions are generally in the presence of foetal calf serum oradded growth factors. However, under normal conditions, in the body,levels of circulating growth factors are too low to be effective fornerve regeneration.

Nerve growth factor (NGF) is one of a family of neurotrophins; otherfamily members include brain-derived neurotrophic factor (BDNF),neurotrophin-3 (NT3) and neurotrophin-4 (NT4; sometimes referred to asNT4/5 or NT5). All of the neurotrophins bind to a common receptor,p75NGFR. Specificity is defined through their interaction with tyrosinereceptor kinases (Trk) the Kd of which interaction is approximately10⁻¹⁰-10⁻¹¹M.

The properties of TrkA are described in WO99/53055. A schematicrepresentation of the TrkA structure is appended as FIG. 1. Thenucleotide sequence and derived amino acid sequence of theimmunoglobulin (Ig)-like binding domain 2 (TrkAIg2) are appended as FIG.2.

NGF binds to TrkA, BDNF and NT4 bind to TrkB and NT-3 binds to TrkC andan alternatively spliced version of TrkA which has a six amino acidinsert VSFSPV (underlined in FIG. 2) in its Ig-like binding domain 2.

The majority of peripheral and spinal nerves require the presence of oneor more of the neurotrophins for survival. Recent studies indicate thatneurotrophic factors play a significant role in helping the developingand adult nervous system survive after axotomy. Before regenerating,neurones need to first survive axotomy. Neurotrophins rescue immature(Diener and Bregman (1994) Neuroreport, 5: 1913) and mature (Shibayamaet al (1998), J Comp Neurol, 390: 102) axotomised central nervous system(CNS) neurones from retrograde cell death. Axotomy of neurones in theperipheral nervous system (PNS) frequently leads to upregulation ofregeneration-associated genes, which assist in regeneration. Onlytransient increases in these genes occur in the CNS after axotomy, closeto the cell body, but not when the lesion is more distal. Prolongedinduction of regeneration-associated genes may be required forregeneration in this situation. Neurotrophins increase the expression ofregeneration associated genes (e.g. c-Jun, GAP-43, Ta1tubulin). Incultured adult dorsal root ganglion cells (DRG), types of axon growth(arborization or elongation) depend on different patterns of geneexpression (Smith and Skene (1997) J Neurosci, 17: 646). BDNF, forinstance, enhances GAP-43, supporting the branching process.

Some researchers have grafted cells that are genetically modified tosecrete growth factors such as NGF at the injury site (Grill et al(1997) Exp Neurol, 148: 444), whilst others have looked at the releaseprofile of NGF, co-encapsulated with ovalbumin, from biodegradablepolymeric microspheres such as those prepared from PLGA 50/50, PLGA85/15, PCL and a blend of PCL/PLGA 50/50 (Cao and Schoichet (1999)Biomaterials, 20: 329). NGF was found to be released and bioactive forat least 3 months.

Other researchers have tried to transplant foetal cells into the site ofinjury in the spinal cord. The remodelling of axonal projections in vivoafter spinal cord injury and transplantation is regulated by theavailability of neurotrophic factors. In the adult, exogenous NGFincreases the growth of axotomised dorsal root axons into the spinalcord (Oudega and Hagg (1996) Exp Neurol, 140: 218; Oudega et al (1994)Exp Neurol, 129: 194). After spinal cord hemisection and foetal cordtransplantation in the adult, the exogenous administration of BDNF, NT3and NT4 increased the amount of supraspinal growth into the foetaltransplant. Ciliary derived neurotrophic factor (CNTF) failed to dothis. BDNF and NT3 also support the regrowth of brainstem fibres intoSchwann cell grafts placed into thoracic level lesions in the adult rat(Xu et al (1995) Exp Neurol, 134: 261). Cells modified to secrete NGFand NT3, transplanted into spinal cord, influence the axonal growth ofspinally projecting neurones (Tuszynski et al (1996) Exp Neurol, 137:157; Grill et al (1997) J Neurosci, 17: 5560), and are associated withan improvement in motor function (Grill et al (1997) J Neurosci, 17:5560).

NT3 and BDNF also induce oligodendrocytic proliferation and myelinationof regenerating axons in the spinal cord after contusion injury (McTigueet al (1998) J Neurosci, 18: 5354).

Recent studies have shown that, in addition to acute injury,neurotrophins may assist regrowth in chronic injury (Ye and Houle (1997)Exp Neurol, 143: 70; Houle et al (1997) Restorative Neurol Neurosci, 10:205; Houle and Ye (1997) Neuroreport, 8: 751).

The prior art described above, whilst indicating the importance ofneurotrophic growth factors in neuronal survival, repair andregeneration, and the possibility of growing neuronal tissue onbiodegradable polymer scaffolds in vitro, leaves open the question ofhow neuronal cells can be efficiently grown under conditions of lowprevailing growth factor concentration. Furthermore, control of the rateand direction of neuronal growth is not addressed.

It is an object of the present invention to provide products and theiruses which are capable of supporting the growth, regeneration and/orrepair of neuronal and other tissues and cells and which do not sufferto such an extent from the problems identified in relation to the priorart.

Accordingly, a first aspect of the invention provides a biocompatible,biodegradable composition for encouraging controlled neuronal growth,regeneration or repair, the composition comprising a scaffold, formedfrom biodegradable and biocompatible material, and a tyrosine receptorkinase (Trk), or a neurotrophin-binding fragment or homologue thereof,located at or adjacent a surface of the scaffold.

The term ‘biodegradable’ as used herein means capable of being brokendown, fragmented and/or dissolved on exposure to physiological orphysiological-type media at pH6.0 to 8.0 and a temperature of 25 to 37°C. The period over which such breaking down, fragmentation and/ordissolution occurs will depend upon the intended application of thecomposition. Typical periods will be less than or about five years, moreoften between one week and one year. The term ‘biocompatible’ as usedherein means that the material to which the term refers, and itsbiodegradation products, are not unacceptably toxic, immunogenic,allergenic or pro-inflammatory when used in vivo. The term ‘scaffold’ asused herein refers to any structure upon, within or through which cellsmay be supported for growth, regeneration or repair.

Preferably, the composition of the invention will include one or moretypes of neurotrophin bound to the Trk or Trk fragment or homologue. Theneurotrophins may be selected from nerve growth factor, brain-derivedneurotrophic factor, neurotrophin-3 and neurotrophin 4.

The Trk of the composition may be TrkA, B or C, an alternatively splicedversion thereof, a pan-Trk (i.e. a Trk which is capable of binding allthe neurotrophins), a functional homologue of a Trk or a combination ofTrk types. Fragments of Trk homologues, and homologues of Trk fragments,are also included. In preferred embodiments, the neurotrophin-bindingfragment of the Trk comprises an immunoglobulin (Ig)-like sub-domain,preferably the Ig-like sub-domain 2 of TrkA (TrkAIg2 or TrkAIg2.6, shownin FIG. 2 as amino acids 22 to 150, with the six amino acids 130 to 135only present in the TrkAIg2.6 splice variant). Alternatively, oradditionally, the neurotrophin-binding fragment of the Trk may compriseboth Ig-like sub-domains of TrkA (TrkAIg1,2). Such fragments of Trk Apreferably also include the proline-rich region. When the Ig-likesub-domain 2 of TrkA is employed, either alone or with the Ig-likesub-domain 1, it preferably includes the amino acid insert VSFSPV(TrkAIg2.6, the insert is shown as amino acids 130 to 135 in FIG. 2).The neurotrophin-binding fragment of the Trk may comprise, or mayconsist of, the entire sequence shown in FIG. 2 (TrkAIg2.6-6His).

When the composition includes one or more types of neurotrophin, it ispreferred that, if TrkA or a neurotrophin-binding fragment thereof isused, the neurotrophin is selected from NGF and NT3. If TrkB or aneurotrophin-binding fragment thereof is used, the neurotrophin ispreferably selected from BDNF and NT4. If TrkC or a neurotrophin-bindingfragment thereof is employed, NT3 is preferred.

In some embodiments of the present invention, the composition alsoincludes one or more extracellular matrix components located at oradjacent a surface of the scaffold. These extracellular matrixcomponents may comprise peptides containing the sequences RGD, YIGSRand/or IKVAV in order to encourage integrin or other cell-surfacereceptor-mediated neurone extension and growth factor responses.

Cells cultured upon predominantly hydrophilic biomaterials such asPLA-PEG-biotin require the additional presence of extracellular matrixmolecules to adhere the cells to the surface.

Such extracellular matrix molecules include collagens, proteoglycans,elastin, hyaluronic acid and glycoproteins such as fibronectin (FN),vitronectin (VN), and laminin (LN). Short peptide domains found alongthese molecules are responsible for interacting with cell-surfaceadhesion receptors known as integrins. Binding of these receptorsfacilitates not only cell adhesion, but also triggers intercellularevents such as migration, spreading and phenotypic expression. Althoughthe whole extracellular molecule can be used in combination with agrowth factor modified surface, intact adhesion molecules typicallyinteract with a wide range of cell types with varying degrees ofspecificity. It may therefore be preferable to employ the short isolatedpeptide sequences, in order to create materials that specificallyinteract with targeted cell types to produce pre-defined responses.

Example integrin-binding peptide sequences include the ubiquitousArginine-glycine-aspartic acid (RGD) sequence, which interacts with mostcell types, and the Isoleucine-lysine-valine-alanine-valine (IKVAV),Leucine-arginine-glutamic acid (LRE), andTyrosine-Isoleucine-glycine-serine-arginine (YIGSR) fragments, which areisolated from laminin and have been demonstrated to facilitate neuronaldevelopment. These peptide sequences may be used in combination, andtheir activity enhanced by using flanking peptide sequences to improvesequence accessibility.

In designing adhesion-peptide-modified surfaces, the surfaceconcentration must be optimised. A minimum density of adhesion ligand isnecessary for cell adhesion and migration, and high densities of peptidewill inhibit cellular migration due to the strength of the adhesion(Huttenlocher, Sandborg, Horwitz (1995) Adhesion in cell migration.Curr. Opin. Cell Biol., 7: 697-706). An intermediate level of attachmentforce is therefore required to induce maximal migration rates (Schense,Hubbell (2000) Three-dimensional migration of neurites is mediated byadhesion site density and affinity. J. Biol. Chem., 275: 6813-6818;Palecek, Loftus, Ginsberg, Lauffenburger, Horwitz (1997) Integrin-ligandbinding properties govern cell migration speed through cell-substratumadhesiveness. Nature, 385: 537-540).

The material of the scaffold is preferably a biodegradable andbiocompatible polymer. The biodegradable and biocompatible polymer maybe selected from: polyhydroxy acids such as polyhydroxybutyric acid,poly (lactic acid), poly (glycolic acid), poly (ε-caproic acid), poly(ε-caprolactone), polyanhydrides, polyorthoesters, polyphosphazenes andpolyphosphates; polysaccharides such as hyaluronic acid; proteins suchas collagen; poly (amino acids); poly (pseudo amino acids); andcopolymers prepared from the monomers of any of these polymers. Polymersof lactic acid or glycolic acid, or copolymers of these monomers, arepreferred. Particularly preferred are block copolymers of any of theabove polymers with a poly(alkylene glycol), such as poly(ethyleneglycol) (PEG). Most preferred are block copolymers of PEG withpoly(lactic acid), poly(glycolic acid) or poly(lactic-co-glycolic) acid.The properties and advantages of these various polymers may be found inWO99/36107.

The Trk or fragment thereof may be located at or adjacent the surface ofthe scaffold, and more preferably at the end of a poly(alkyleneglycol)chain when block copolymers comprise the scaffold, by any meanscompatible with the biocompatible, biodegradable material and the Trk.Such means may include covalent attachment, adsorption or physicalentrapment. It is preferred, however, that the Trk or fragment isattached to or adjacent the surface by means of one or more specificmolecular interactions. By ‘specific molecular interactions’ is meantinteractions between two or more binding components with at least100-fold higher affinity, preferably at least 500-fold, at least1000-fold or at least 2000-fold higher affinity, than that of theinteraction between one of those binding components and other moleculeswhich it may encounter, e.g. in cell culture or in vivo. The one or morespecific molecular interactions which attach the Trk to or adjacent thesurface of the scaffold preferably take place between one or more anchormolecules bound to or adjacent the scaffold surface and one or more tagmolecules bound to the Trk or fragment.

The anchor and tag molecules may be the same or different. In certainembodiments, the anchor is an antibody or fragment thereof and the tagis the corresponding antigen or hapten, or vice versa. Preferably, thetag is biotin and the anchor is avidin or streptavidin, or vice versaMost preferably, an adapter molecule is also used which is capable ofsimultaneously binding to both the tag and the anchor. In such a case,both the tag and the anchor may be the same. In preferred embodiments,both the tag and the anchor are biotin and the adapter is avidin orstreptavidin (avidin and streptavidin have a valency of 4 in theirbinding to biotin).

It will be appreciated that only one specific molecular interaction needbe employed in the attachment of a Trk or fragment to or adjacent thesurface of the scaffold in order for the composition to benefit from theadvantages associated with specific molecular interactions. Thus, anyother molecular interactions (e.g. between the anchor and an adaptermolecule when the tag binds to the adapter by means of a specificmolecular interaction) need not be specific.

Methodologies suitable for the covalent attachment of the anchormolecule to or adjacent the scaffold surface and of the tag molecule tothe Trk or fragment are well known in the art and reference may be madeto WO99/36107 and references cited therein. The composition of thepresent invention preferably has a tubular scaffold, the regeneration ofthe neurones preferably taking place along the lumens of the tubularstructure. The Trk or fragment may be located on the luminal wall byflowing a solution of the Trk or fragment through a scaffold previouslytreated so as to be capable of binding the Trk or fragment. Thus, in thecase of specific molecular interactions, the scaffold may previouslyhave been treated such that the luminal walls are labeled with anchormolecules, the Trk in solution being labeled with tag molecules. If anadapter molecule is employed, this is presented to the scaffold beforethe tagged Trk.

Any additional components to be located on or adjacent the scaffoldsurface may be attached in the same manner as the Trk or fragments. Whenthe composition includes one or more neurotrophins, these are introducedto the scaffold either bound to the Trk or fragment, or as a separatestep following prior location of the Trk or fragment. In each case wherea Trk, tag, adapter, anchor, neurotrophin or any other component of thecomposition is introduced to the scaffold in solution, it is generallyuseful to introduce an excess to ensure adequate loading of bindingsites. The excess, some of which may, of course, have becomenon-specifically bound to the scaffold or other components, may then beflushed out. The preparation of surfaces in a manner similar to thosewhich may be used in the present invention is described in WO99/36107.In particular, the patterning of Trk on or adjacent the surface of thescaffold may be achieved using methods analogous to those used inWO99/36107.

The composition may also include growing, regenerating or repairingnerve cells, or nerve cell progenitors or pluripotent stem cells.

The compositions of the present invention have the advantage that theyallow a ready supply of neurotrophins to be made available for neuronaluptake. The neurotrophins are non-covalently bound to the scaffold andhence are releasable for use by neurones. This provides neurotrophicsupport for the neurones in a way not envisaged previously. Furthermore,and particularly in those embodiments where a spatially arranged, orpatterned, location of Trk or fragments is employed, a directionalneuronal extension may be achieved. The composition of the invention maybe used, either in vitro or in vivo both as a sequesterer ofneurotrophins for subsequent supply to neurones (in which case thecomposition may be employed with few or no neurotrophins boundinitially) and as a source of neurotrophins for neurones (in which casethe composition may include a higher proportion of neurotrophin-boundTrk molecules or fragments).

The levels of circulating neurotrophins are too low to support survival.Neurotrophins are normally released from innervated tissues and areinternalised by neurones after binding to Trk receptors. This complex isthen transported to the cell body where it is thought to exert its cellsurvival effects. In order to regenerate nerves in vivo it will benecessary to provide a local supply of neurotrophins. The presentinvention allows the neurotrophins to be supplied non-covalently boundto the scaffold via the Trk molecule or fragment.

In a second, and related, aspect of the invention there is provided thecomposition of the first aspect for use in therapy.

In a third aspect there is provided the use of a Trk, or aneurotrophin-binding fragment or homologue thereof, in the preparationof a medicament for encouraging nerve growth, regeneration or repair,the medicament comprising a scaffold formed from a biodegradable andbiocompatible material, and the Trk or fragment or homologue beinglocated at or adjacent a surface of the scaffold.

The invention also provides, in a fourth aspect, a method of encouragingnerve growth, regeneration or repair, the method comprising contacting acomposition according to the first aspect of the invention with a sourceof neurotrophins so as to form Trk-neurotrophin complexes on or adjacentthe surface of the scaffold, contacting the composition with a stemcell, nerve progenitor cell, neuronal cell or tissue and allowing thestem cell, nerve progenitor cell, neuronal cell or tissue to grow,regenerate or repair upon or adjacent the surface of the scaffold.

000The method of the fourth aspect may be carried out in vivo or invitro. The source of neurotrophins may comprise the innervated site intowhich the composition is placed in an in vivo embodiment of the method.More preferably however, in both in vivo and in vitro embodiments, thesource of neurotrophins comprises a solution of neurotrophins which isflowed through or over the surface of the scaffold having the locatedTrk or fragment. The method is preferably used for the regeneration ofsevered nerves in vivo.

The invention also provides a stem cell, nerve progenitor cell, neuronalcell or tissue obtained or obtainable by a method according to thefourth aspect of the invention.

In a fifth, and related, aspect, the present invention provides a methodof transplanting stem cells, nerve progenitor cells, nerve cells ortissue, the method comprising taking a sample of donor stem cells, nerveprogenitor cells or nerve cells from a suitable donor culture orsubject; growing, regenerating or repairing the donor cells in contactwith a composition according to the first aspect of the invention havingTrk-neurotrophin complexes on or adjacent the surface of the scaffold;and placing the donor cells and composition into a recipient subject inneed of such donor cells.

The donor and recipient subjects may be the same (i.e. an autologousgraft) or different (i.e. an heterologous graft) individuals.

The compositions, methods and uses of the invention described so faravoid, at least in part, several of the shortfalls associated with priorart technology in this field. Such shortfalls include, in the case ofperipheral nerve repair using autograft material, donor site morbidity,inadequate return of function and aberrant regeneration. The use ofsynthetic biodegradable polymers in conjunction with Schwann cells islimited by the need for additional rounds of cell culture and by thenecessity of the incorporation of cells into the site to be treated.Nerve transplants are to be avoided if possible since they may exposethe patient to an increased risk of variant-Creutzfelt-Jakob disease.The prior art relating to biodegradable polymer surfaces presentingRGD-type peptides provides a method of directing cell spreading andregeneration but does not address how vital growth factors can beprovided, especially in an in vivo setting. The growth factors requiredby growing, repairing and/or regenerating neurones need to be availableat the neuronal cell surface for uptake. Using the present invention, itis possible to form patterns of Trk or fragments on or adjacent ascaffold surface and thereby to hold one or more of a variety ofneurotrophins for presentation to growing neurones.

In a sixth aspect of the present invention there is provided abiocompatible, biodegradable composition for controlled release of a Trkor fragment or homologue thereof, the composition comprising areservoir, formed from a biodegradable and biocompatible material, and aTrk, or a neurotrophin-binding fragment or homologue thereof, intimatelyassociated with the reservoir and/or located at or adjacent a surface ofthe reservoir.

The reservoir may have any of the preferred features of the scaffolddescribed above. The Trk may be bound to the reservoir surface asdescribed above. The composition may contain both surface-bound Trk orfragments and Trk or fragments embedded within the material of thereservoir. Such a mixed system may provide greater flexibility in thecontrol of Trk release rates. The composition may be suitable for invitro and/or in vivo use.

The invention also provides a composition according to the sixth aspect,for use in therapy.

In a seventh and related aspect, the invention provides the use of aTrk, or a neurotrophin-binding fragment or homologue thereof in thepreparation of a controlled release medicament for the treatment of acondition associated with elevated neurotrophin levels, the medicamentcomprising a reservoir formed from a biodegradable and biocompatiblematerial, and the Trk or fragment or homologue being intimatelyassociated with the reservoir and/or located at or adjacent a surface ofthe reservoir.

The invention also provides a method of treatment of a conditionassociated with elevated neurotrophin levels in a subject, the methodcomprising the administration to the subject of a composition accordingto the sixth aspect of the invention.

The condition to be treated may be Alzheimer's disease or may be a paindisorder. The pain may be a symptom of idiopathic sensory urgency (ISU),interstitial cystitis, arthritis, shingles, peripheral inflammation,chronic inflammation, an oncological condition or postherpeticneuralgia.

In an eighth aspect, the invention provides a biocompatible,biodegradable composition for encouraging controlled growth,regeneration or repair of biological tissue or cells, the compositioncomprising a scaffold, formed from biodegradable and biocompatiblematerial, and a receptor for a growth factor, or a growth factor-bindingfragment or homologue thereof, located at or adjacent a surface of thescaffold.

The growth factor may be a neurotrophin.

Furthermore, in a ninth aspect, the invention provides a biocompatible,biodegradable composition for encouraging controlled growth,regeneration or repair of biological tissue or cells, the compositioncomprising a scaffold, formed from biodegradable and biocompatiblematerial, and a growth factor, or a functional fragment or homologuethereof, located at or adjacent a surface of the scaffold. The growthfactor, which may be a neurotrophin, may be covalently or non-covalentlybound to the scaffold. The invention also provides a compositionaccording to the ninth aspect, for use in therapy.

The invention will be now described in more detail by way of exampleonly and with reference to the appended drawings, of which:

FIG. 1 shows a schematic representation of the TrkA structure;

FIG. 2 provides the nucleotide and derived amino acid sequence ofTrkAIg2, including the N-terminal six-His tag and the six amino acidinsert VSFSPV (underlined);

FIG. 3 illustrates the results of an experiment looking at the in vitroeffects of Trk- and NGF-modified surfaces on neurite growth;

FIG. 4 illustrates, schematically, a protocol for the production of atissue regeneration scaffold comprising either patterned channels ofligand or tubes lined with ligands;

FIG. 5 shows a simplified, partial cross-sectional structuralrepresentation of a composition according to the present invention; and

FIG. 6 illustrates how the composition of the present invention may beused to encourage neuronal growth and extension.

EXAMPLE 1 Structure of TrkAIg₂ and TrkAIg2.6

TrkA and isolated domains thereof are further described in WO99/53055,the disclosure of which is incorporated by reference. The accompanyingFIG. 1 illustrates its structure schematically (also Robertson et al(2001) BBRC, 282: 131). The filled circles represent glycosylationsites. TrkAIg2 is defined in this example as including Ig-like subdomain2 and the proline rich region. The sequence (TrkAIg2.6-6His) shown inFIG. 2 shows the nucleotide sequence and derived amino acid sequence ofTrkAIg2 with 6×His tag. Sequence from human TrkA is in bold, 6 aminoacid insert variant is underlined. This sequence includes the human TrkAsequence (amino acids 22 to 150) and a flanking sequence from the pET15bvector (amino acids 1 to 21) which also codes for an N-terminal 6×Histag. The vector sequence (codons 452 to 468, FIG. 2) also provides for astop codon. The putative extracellular domain of human TrkA is taken tobe either 375 or 381 amino acids long depending on whether the 6 aminoacid insert VSFSPV is present.

It has recently been shown that a protein consisting of the twoimmunoglobulin-like domains and proline-rich region alone are able tobind NGF with a similar affinity to that of the complete extracellulardomain (Holden et al (1997) Nature Biotechnology, 15: 668). This regionis defined here as TrkAIg1,2. In addition, it has been found that aneven smaller domain of TrkA, referred to as TrkAIg2 (shown in FIG. 2 asamino acids 22 to 150) able to bind NGF with a similar affinity to thecomplete extracellular domain or the TrkAIg1,2 region and is thusprimarily responsible for the binding properties of these largerentities. TrkAIg2 which contains the six amino acid insert VSFSPV, asshown in FIG. 2 as amino acids 130 to 135, is referred to here asTrkAIg2.6.

EXAMPLE 2 Neuronal Growth Enhancement by Immobilisation of NGF Using Trk

This study demonstrates the feasibility of using a Trk fragment toimmobilise NGF to a biomaterial surface and thus provide a localisedenvironment to stimulate peripheral nerve regeneration. Experiments wereperformed using PLA-PEG-biotin as a base material, which has previouslybeen demonstrated to enable facile surface patterning of ligands tospatially control tissue regeneration (WO99/36107). In this Example, theTrk fragment used was the 6-His tagged version of TrkIgA2.6(TrkIgA2.6-6His).

PC12 cells were grown in RPMI-1640 media, supplemented with 10% horseserum, 5% foetal calf serum, antibiotic/antimycotic, and L-glutamine, ata density of 2-5×10⁵ cells/ml, Each T75 flask containing between 10-20ml of media.

As PC12 cells require surface attachment in order to enable neuriteextension, their non-adherence to tissue culture plastic means thatsurfaces must be precoated with extracellular matrix substrates such ascollagen or laminin. T-75 flasks and 24 well plates were collagen coatedby leaving a 0.01% collagen Type IV solution in distilled sterile wateron the surface for two hours (10 ml and 0.5 ml respectively) and thenair drying overnight.

Prior to the TrkAIg2.6 neurite extension studies, PC-12 cells grown uponcollagen-coated flasks were primed with NGF (50 ng/ml) for 5 days, freshmedia and NGF being added every 24 hours.

TrkA2.6-6His (in 20 mM Sodium Phosphate buffer pH 8.0, 100 mM SodiumChloride and 10% glycerol) was prepared using the method of WO99/53055and pending application number PCT/GB02/04214 and a Sigma kit was usedto biotinylate using standard procedures. A stock solution ofapproximately 250 μg/ml was prepared.

PLA-PEG-biotin coated Iwaki Non-Treated 24 well plates were preparedusing 0.25 ml of 2 mg/ml polymer dissolved in 2,2,2-Trifluoroethanol(TFE), dropcast onto well plates & dried in oven at 60° C. for 1 hour.Plates were then washed in PBS & stored in a refrigerator overnight.Three test plates were prepared for each batch of biotinylatedTrkAIg2.6.

Avidin was attached to the PLA-PEG-biotin-coated plates using 0.5 ml ofa 500 μg/ml solution in distilled water for 45 mins at 37° C., beforeagain washing the plates with PBS.

1 ml of the biotinylated TrkA prepared above was then added to theplates at a conc. of between 0-250 μg/ml in distilled water for 1 hourat 37° C. Plates were then washed with PBS. 0.5 ml of 0-50 ng/l (=0-1μl/ml) of NGF in PBS was then added for 45 mins at 37° C. before a finalPBS wash. This was then followed by coating of all wells with 0.25 ml of0.0025% Collagen Type IV in distilled water for 1 hour before washing.Control wells contained NGF media in the presence of PLA-PEG-Biotin or0.01% collagen, but no TrkAIg2.6.

Cells (passage 18) were seeded at 2.0×10⁴ cells/ml per well. The mediawas replaced with a fresh supply (containing 0, 0.1 or 1 μl NGF) after24 hours.

After allowing the cells to attach and extend neurites over a 48-hourperiod in an incubator set at 37° C./5% CO₂, the media was aspirated toremove any loosely adherent cells and again replaced. Images were thentaken using a Nikon Eclipse TS100 microscope and DN100 digital camera at× magnification with a 0.45× C-mount. Process length and cell numberwere measured using Leica Qwin image analysis software.

FIG. 3 shows neurite extension from the PC12 nerve cell line followingculture upon a range of Trk-, and subsequently, NGF-modified surfaces.The data shows an increase in neurite extension with increasing amountsof surface-immobilised NGF, and that an optimal Trk concentrationappears to be within the range used.

This study illustrates the ability to induce neurite outgrowth fromcells cultured upon modified PLA-PEG-biotin surfaces, by presenting NGFusing a receptor fragment to attach the growth factor to the surface.Extracellular matrix molecules are also preferably presented at thesurface in order to enhance cell surface attachment.

Within this data, a Trk concentration-dependant effect can be seen ateach different NGF concentration and it appears that an optimum Trkconcentration exists. For TrkAIg2.6, this may be between 2.5 and 250μg/ml, depending on experimental conditions, and may be around 25 μg/ml.The decrease in neurite outgrowth at the higher concentration may be dueto toxicity effects or increased competition for NGF between immobilisedreceptor fragments and the cells themselves.

EXAMPLE 3 A Nerve Regeneration Scaffold

FIG. 4 shows schematically how a scaffold suitable for a compositionaccording to the invention may be generated. The scaffold may befabricated to comprise patterned channels of ligand (A) or to comprisetubes which have a patterned lining of ligand (B). FIG. 5 showsschematically how a composition according to the invention may beassembled. Briefly, the polymer scaffold or matrix of FIG. 4 (polyester,such as poly(lactic acid), poly(lactic-co-glycolic) acid or blockcopolymers of these polyesters with PEG) is patterned (as described inWO99/36107) with biotin molecules. Avidin is then introduced to thescaffold to produce an avidin-patterned surface. Biotin-labelledTrkAIg2.6 is then passed through the scaffold; the biotin labels bind tounoccupied binding sites on the avidin molecules and thus produce aTrk-patterned surface. Finally, neurotrophins are introduced; these bindto the TrkAIg2.6 and thus produce a neurotrophin-patterned,biodegradable polymer scaffold.

In the example shown in FIG. 5 (and more clearly in FIG. 6), thescaffold has a structure comprising a number of tubes or conduits, ofwhich one is shown. Nerve cells are able to grow along the lumen of thetube/conduit, obtaining neurotrophins from the prepared surface as theydo so. In FIG. 5, it can be seen that the neurotrophins are taken up bythe nerve cells by means of the Trk molecules expressed on the surfaceof the cells. The composition of the invention may be used, inparticular, in nerve regeneration following acute spinal injury, acuteperipheral injury and chronic injury.

EXAMPLE 4 A controlled Release Formulation of Trk

An implantable polymeric (poly(lactic-co-glycolic)acid) reservoir ofTrkAIg2.6 was prepared as described in Example 3 in relation to theneuronal repair scaffold but with the exclusion of the final step ofadding neurotrophins. The reservoir was implanted in an in vivo model ofneuropathic pain. Prolonged release of Trk and resulting analgesia wereobserved.

1. A biocompatible, biodegradable composition for encouraging controlled neuronal growth, regeneration or repair, the composition comprising a scaffold, formed from biodegradable and biocompatible material, and a tyrosine receptor kinase (Trk), or a neurotrophin-binding fragment or homologue thereof, located at or adjacent a surface of the scaffold.
 2. A composition according to claim 1 wherein the Trk is TrkA, B or C, an alternatively spliced version thereof, a pan-Trk, a functional homologue of a Trk or a combination of Trk types.
 3. A composition according to claim 1 wherein the fragment of the Trk comprises an Ig-like sub-domain.
 4. A composition according to claim 3 wherein the fragment comprises Ig-like sub-domain 2 of TrkA.
 5. A composition according to claim 4 wherein the Ig-like sub-domain 2 includes the amino acid insert VSFSPV.
 6. A composition according to claim 5 wherein the fragment comprises the sequence shown in FIG. 2 or a functional homologue thereof.
 7. A biocompatible, biodegradable composition for encouraging controlled neuronal growth, regeneration or repair, the composition comprising a scaffold, formed from biodegradable and biocompatible material and a tyrosine receptor kinase (Trk), or a neurotrophin-binding fragment or homologue thereof located at or adjacent a surface of the scaffold and including one or more types of neurotrophin bound to the Trk or fragment or homologue.
 8. A composition according to claim 7 wherein the neurotrophin is selected from NGF, BDNF, NT3 and NT4.
 9. A composition according to claim 1 or claim 7 including one or more extracellular matrix components located at or adjacent a surface of the scaffold.
 10. A composition according to claim 9 wherein the extracellular matrix components include collagen.
 11. A composition according to claim 9 wherein the extracellular matrix components comprise peptides containing the sequences RGD, YIGSR and/or IKVAV.
 12. A composition according to claim 1 or claim 7 wherein the material of the scaffold is a biodegradable and biocompatible polymer.
 13. A composition according to claim 12 wherein the polymer is selected from polyhydroxy acids, polysaccharides, poly (amino acids), poly (pseudo amino acids), and copolymers prepared from the monomers of any of these polymers.
 14. A composition according to claim 13 wherein the polymer is a block copolymer with a poly (alkylen glycol).
 15. A composition according to claim 14 wherein the polymer is a block copolymer of poly (ethylene glycol) with poly (lactic acid), poly (glycolic acid) or poly (lactic-co-glycolic) acid.
 16. A composition according to claim 1 or claim 7 wherein the Trk or fragment or homologue is located at or adjacent the surface of the scaffold by means of one or more specific molecular interactions and the material of the scaffold is a biodegradable and biocompatible polymer.
 17. A composition according to claim 16 wherein the one or more specific molecular interactions take place between one or more anchor molecules bound to or adjacent the scaffold surface and one or more tag molecules bound to the Trk or fragment or homologue.
 18. A composition according to claim 17 wherein a tag molecule is biotin and an anchor molecule is avidin or streptavidin, or vice versa.
 19. A composition according to claim 17 wherein an adapter molecule is used which is capable of simultaneously binding to both the tag and the anchor.
 20. A composition according to claim 19 wherein both the tag and the anchor are biotin and the adapter is avidin or streptavidin.
 21. A composition according to claim 1 or claim 7 wherein the scaffold is tubular in shape.
 22. A composition according to claim 1 or claim 7 wherein the Trk is present at a concentration of around 2.5 to 250 μg/ml.
 23. A biocompatible, biodegradable composition for encouraging controlled growth, regeneration or repair of biological tissue or cells, the composition comprising a scaffold, formed from biodegradable and biocompatible material, and a receptor for a growth factor, or a growth factor-binding fragment or homologue thereof, located at or adjacent a surface of the scaffold.
 24. A composition according to claim 23 wherein the growth factor is a neurotrophin.
 25. A composition according to claim 1, 7, or 23 wherein the Trk or other growth factor receptor is patterned at or adjacent the surface of the scaffold so as to provide directional control of growth, regeneration or repair of the neuronal or other biological tissue or cells. 26-27. (canceled)
 28. A method of encouraging nerve growth, regeneration or repair, the method comprising contacting a composition according to any of claims 1 or 7 with a source of neurotrophins so as to form Trk-neurotrophin complexes on or adjacent the surface of the scaffold, contacting the composition with a stem cell, nerve progenitor cell, neuronal cell or tissue and allowing the stem cell, nerve progenitor cell, neuronal cell or tissue to grow, regenerate or repair upon or adjacent the surface of the scaffold.
 29. A method of transplanting stern cells, nerve progenitor cells, nerve cells or tissue, the method comprising taking a sample of donor stem cells, nerve progenitor cells, or nerve cells from a suitable donor culture or subject; growing, regenerating or repairing the donor cells in contact with a composition according to any of claims 1 or 7 having Trk-neurotrophin complexes on or adjacent the surface of the scaffold; and placing the donor cells and composition into a recipient subject in need of such donor cells.
 30. A biocompatible, biodegradable composition for controlled release of a Trk or fragment or homologue thereof, the composition comprising a reservoir, formed from a biodegradable and biocompatible material, and a Trk, or a neurotrophin-binding fragment or homologue thereof, intimately associated with the reservoir and/or located at or adjacent a surface of the reservoir.
 31. (canceled)
 32. A method of treating a condition associated with elevated neurotrophin levels, comprising administering a composition according to claim 30 to a subject in need thereof.
 33. The method of claim 32 wherein the condition to be treated is Alzheimer's disease or a pain disorder.
 34. The method of claim 33 wherein the pain disorder is associated with idiopathic sensory urgency, interstitial cystitis, arthritis, shingles, peripheral inflammation, chronic inflammation, an oncological condition or postherpetic neuralgia.
 35. A stem cell, nerve progenitor cell, neuronal cell or tissue obtained a method according to claim
 28. 36. A biocompatible, biodegradable composition for encouraging controlled growth, regeneration or repair of biological tissue or cells, the composition comprising a scaffold, formed from biodegradable and biocompatible material, and a growth factor, or a functional fragment or homologue thereof, located at or adjacent a surface of the scaffold.
 37. (canceled)
 38. A composition according to claim 2 wherein the fragment of the Trk comprises an Ig-like sub-domain.
 39. A composition according to claim 38 wherein the fragment comprises Ig-like sub-domain 2 of TrkA.
 40. A composition according to claim 39 wherein the Ig-like sub-domain 2 includes the amino acid insert VSFSPV.
 41. A composition according to claim 40 wherein the fragment comprises the sequence shown in FIG. 2 or a functional homologue thereof.
 42. A composition according to claim 38 including one or more types of neurotrophin bound to the Trk or fragment or homologue.
 43. A composition according to claim 10 wherein the extracellular matrix components comprise peptides containing the sequences RGD, YIGSR and/or IKVAV.
 44. A composition according to claim 43 wherein the material of the scaffold is a biodegradable and biocompatible polymer.
 45. A composition according to claim 44 wherein the polymer is selected from polyhydroxy acids, polysaccharides, poly (amino acids), poly (pseudo amino acids), and copolymers prepared from the monomers of any of these polymers.
 46. A composition according to claim 45 wherein the polymer is a block copolymer with a poly (alkylen glycol).
 47. A composition according to claim 46 wherein the polymer is a block copolymer of poly (ethylene glycol) with poly (lactic acid), poly (glycolic acid) or poly (lactic-co-glycolic) acid.
 48. A composition according to claim 43 wherein the Trk or fragment or homologue is located at or adjacent the surface of the scaffold by means of one or more specific molecular interactions.
 49. A composition according to claim 48 wherein the one or more specific molecular interactions take place between one or more anchor molecules bound to or adjacent the scaffold surface and one or more tag molecules bound to the Trk or fragment or homologue.
 50. A composition according to claim 49 wherein a tag molecule is biotin and an anchor molecule is avidin or streptavidin, or vice versa.
 51. A composition according to claim 49 wherein an adapter molecule is used which is capable of simultaneously binding to both the tag and the anchor.
 52. A composition according to claim 51 wherein both the tag and the anchor are biotin and the adapter is avidin or streptavidin.
 53. A composition according to claim 43 wherein the scaffold is tubular in shape.
 54. A composition according to claim 43 wherein the Trk is present at a concentration of around 2.5 to 250 μg/ml. 